This is an application for the second renewal of a project which has led to the initial description of hairpin ribozymes and the subsequent development of catalytic RNA specific for HIV-1, which is currently being evaluated as a therapeutic for AIDS. The overall objective of this proposal is to further develop this class of hairpin ribozymes by extending the sequence requirements at the site of cleavage. The initial hairpin ribozyme modeled from the naturally occurring catalatic RNA of tobacco ringspot virus (sTRSV) has a strong preference for cleavage at sites just before a GUC in the target RNA. During the current funding period, the investigator has identified two new ribozymes in the hairpin family which are capable of efficiently cleaving substrate with a GUA after the cleavage site. These originated from the negative strands of the satellite RNAs from chicory yellow mottle virus (sCYMV1) and from arabis mosaic virus (sArMV), and in this proposal, the investigators will develop ribozymes suitable for cleavage of HIV-1 RNA containing these GUA target sites. Specifically, the investigators will determine substrate targeting rules for the sCYMV1 and sARMV ribozymes and pursue the reasons for GUC or GUA specificity in the various hairpin ribozymes. The investigators propose to modify the ribozymes having GUA specificity and, in an attempt to enhance activity and increase stability, they will develop and optimize these ribozymes for cleavage of HIV-1 sequences. By expanding repertoire of ribozymes capable of targeting HIV-1, chances of overcoming viral mutagenic adaptation would be enhanced.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI029870-08
Application #
2390345
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1990-04-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
8
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Northern Illinois University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
De Kalb
State
IL
Country
United States
Zip Code
60115
Lian, Y; De Young, M B; Siwkowski, A et al. (1999) The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA. Gene Ther 6:1114-9
Shippy, R; Siwkowski, A; Hampel, A (1998) Mutational analysis of loops 1 and 5 of the hairpin ribozyme. Biochemistry 37:564-70
Siwkowski, A; Humphrey, M; De-Young, M B et al. (1998) Screening for important base identities in the hairpin ribozyme by in vitro selection for cleavage. Biotechniques 24:278-84
DeYoung, M B; Siwkowski, A; Hampel, A (1997) Determination of catalytic parameters for hairpin ribozymes. Methods Mol Biol 74:209-20
Hampel, A; DeYoung, M B; Galasinski, S et al. (1997) Design of the hairpin ribozyme for targeting specific RNA sequences. Methods Mol Biol 74:171-7
Siwkowski, A; Shippy, R; Hampel, A (1997) Analysis of hairpin ribozyme base mutations in loops 2 and 4 and their effects on cis-cleavage in vitro. Biochemistry 36:3930-40
Siwkowski, A (1997) T7 transcript length determination using enzymatic RNA sequencing. Methods Mol Biol 74:91-7
Vinayak, R; Andrus, A; Sinha, N D et al. (1995) Assay of ribozyme-substrate cleavage by anion-exchange high-performance liquid chromatography. Anal Biochem 232:204-9
Yu, M; Poeschla, E; Yamada, O et al. (1995) In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1. Virology 206:381-6
Altschuler, M; Tritz, R; Hampel, A (1992) A method for generating transcripts with defined 5' and 3' termini by autolytic processing. Gene 122:85-90